Foot and Mouth Disease (FMD)
FMD is a highly contagious and economically devastating disease of cloven-hoofed animals (Artiodactyla), affecting domesticated ruminants, pigs and a large number of wildlife species.
FMD is widely distributed throughout the world. Developed regions such as the continents of North and Central America and Antarctica, and countries such as Australia and New Zealand are free from disease while FMD is endemic in many developing countries such as those in sub-Saharan Africa, the Middle East, southern Asia, Southeast Asia and South America. There are also some areas of the world which are normally free from disease, such as Europe where FMD was eradicated in 1989 and where vaccination has ceased since 1991. However, there have been occasional incursions of disease such as the 2001 UK/EIRE/France/Netherlands epidemic due to a PanAsian O strain (Knowles et al., (2001) Veterinary Record. 148. 258-259) and the 2007 UK outbreak of serotype O1 BFS/1967.
The causal agent of FMD is Foot-and-Mouth Disease Virus (FMDV), a positive sense, single stranded RNA virus of the Picornaviridae family. FMDV exists as seven antigenically distinct serotypes namely A, O, C, Asia 1 and South African Territories (SAT) 1, 2 and 3, with numerous subtypes within each serotype.
Translation of the single-stranded RNA yields a polyprotein that is subsequently processed by virus-encoded proteases to produce the structural and non-structural proteins required for virus assembly and replication. The Leader (L) protease cleaves itself in cis or in trans at its C terminus from the P1-2A capsid precursor. The 2A protease cleaves itself at its C terminus to release P1-2A from P2. Processing of the P1-2A is effected by the 3C protease to produce the capsid proteins 1AB (also known as VP0), 1C (VP3) and 1D (VP1). In the virion, cleavage of 1AB occurs to produce 1A (VP4) and 1B (VP2).
FMDV Vaccines
Conventional vaccines against FMD consist of whole virus virions that have been chemically inactivated, normally by use of an aziridine such as binary ethyleneimine (BEI).
Inactivated whole virus vaccines play a key role in campaigns to control and eradicate FMD. However, vaccines produced from viral tissue culture are associated with the risk of virus release during vaccine production. There is also the risk of improper inactivation of the virus which has the potential to lead to vaccine-related FMD outbreaks.
In order to reduce these risks, the possibility of using empty capsid-like particles of FMDV has been considered. Such particles comprise the structural proteins of FMDV but are non-replicative and non-infectious because they have no RNA genome. As the external structure of the empty capsids should be the same as the wild-type virus, empty capsids should be similarly antigenic and immunogenic.
Several attempts have been made to produce empty FMD capsid particles, but there have been recurring problems associated with yield and stability of the product.
A vaccinia virus expression system has been used to express a P1-2A-3C cassette (Abrahams et al (1995) J. Gen Virol. 76:3089-3098). It was found that constitutive expression of the cassette was unsuccessful but vv/FMDV recombinants could be isolated when the cassette was placed under the control of the bacteriophage T7 promoter. However, such a system could not be used for prolonged expression of empty capsids because after time, the toxicity of the P1-2A-3C cassette would prevail. There is also the issue that constant T7 Pol expression would be needed to drive production of the P1-2A-3C. It may be possible to achieve this at small scale in tissue culture, but it would be not be possible to extrapolate this to a manufacturing scale. Moreover, products produced in a vaccinia system are not commercially viable for a medical or veterinary application.
Li et al (2008) (PLoS ONE 28:3(5) e2273) report the expression of FMDV virus capsid proteins in a silkworm-baculovirus expression system. Recombinant virus expressing the intact coding regions of P1-2A and 3C were used to inoculate silkworms and subsequently the haemolymph collected from the dying silkworms. It was shown that a preparation of these “expressed antigens” caused an anti-FMDV-antibody response in cattle. However, the nature of the “expressed antigens” is entirely unclear, and the authors appear to assume it is a “subunit vaccine” as opposed to an empty capsid.
Cao et al ((2009) Veterinary Microbiology 137:10-17) describes a recombinant baculovirus system which simultaneously expresses the genes for the P12A and 3C proteins of FMDV from individual promoters. It was shown by Western blotting that the capsid proteins were processed to some extent by 3C protease and that empty capsid particles could be observed by immunoelectron microscopy. Immunisation with a crude extract of empty capsid did produce an immune response, but the levels of FMDV-specific antibodies and neutralising antibodies were lower that the conventional inactivated vaccine. It is predicted that this is due to lower levels of empty capsid particles. It is concluded that further studies are needed to improve the amount of protein expression and empty capsid assembly in insect cells.
There is thus a need for an improved method for producing empty virus capsids which produces an immunogenic, stable product at a reasonable yield.